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Real‐time measurements of dark substrate catalysis
Author(s) -
Xie Dong,
Suvorov Leonid,
Erickson John W.,
Gulnik Sergei
Publication year - 1999
Publication title -
protein science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.353
H-Index - 175
eISSN - 1469-896X
pISSN - 0961-8368
DOI - 10.1110/ps.8.11.2460
Subject(s) - cleavage (geology) , substrate (aquarium) , chemistry , protease , kinetics , catalysis , enzyme kinetics , enzyme , peptide , stereochemistry , combinatorial chemistry , chromatography , materials science , active site , biochemistry , biology , physics , ecology , quantum mechanics , fracture (geology) , composite material
We have developed a novel procedure to monitor the real‐time cleavage of natural unmodified peptides (dark substrates). In the competition‐based assay, the initial cleavage rate of a fluorogenic peptide substrate is measured in the presence of a second substrate that is not required to exhibit any optical property change upon cleavage. Using a unique experimental design and steady‐state enzyme kinetics for a two‐substrate system, we were able to determine both K m and k cat values for cleavage of the dark substrate. The method was applied to HIV‐1 protease and to the V82F/I84V drug resistant mutant enzyme. Using two different substrates, we showed that the kinetic parameters derived from the competition assay are in good agreement with those determined independently using standard direct assay. This method can be applied to other enzyme systems as long as they have one substrate for which catalysis can be conveniently monitored in real time.